The functions of the mammalian heat shock transcription factors (Hsfs) are becoming increasingly clear with the recent generation of mice lacking the hsfl or hsf2. The results generated from analyses of these animal models are striking and indicative of the role of Hsfs in apoptosis, differentiation and development as well as maintenance of tissue homeostasis. To understand the functions of Hsfs in vivo, we have generated hsfl and hsf2 knockout mice and are now working to create mice that do not express hsf4. Disruption of hsfl illuminates its importance for regulation of apoptosis and control of thermotolerance and female sterility. In contrast, the hsf2 knockout mouse exhibits defects in development of the central nervous system and reduction in spermatogenesis. We further show evidence that deletion of both hsfl and hsf2 leads to synergistic effects and defects that are more severe than those manifested in individual hsf-deficient mice. These defects are associated with brain and behavioral abnormalities, and complete disruption of spermatogenesis and male sterility, as well as others. Compared to Hsfl and Hsf2, relatively little is known about the only remaining member of the mammalian Hsf family, Hsf4. Several features distinguish Hsf4 from other Hsfs. It is expressed as two alternatively spliced variants with one form being a transcriptional activator and the other a transcriptional repressor and these two variants are differentially expressed in different tissues. We show that Hsf4 is phosphorylated and a novel phosphatase (named DSPH4) containing signature motifs of the dual specificity tyrosine/serine/threonine phosphatases has been identified that binds to Hsf4 and modulates its phosphorylation status, DNA binding ability and nuclear localization. This represents a possible mechanism for the regulation of Hsf4 activity by extracellular signals. Because Hsfl and Hsf2 possess distinct functions, regulatory mechanisms, and target genes, but show some functional interdependency, it is reasonable to postulate that Hsf4 will also have some overlapping function with Hsfl and Hsf2, but also its own unique roles in maintaining cellular homeostasis. The studies outlined in this grant application are directed to understand the regulation of Hsf4 at the molecular level and to examine the function of Hsf4 not only alone, but also in coordination with Hsfl and Hsf2 functions in vivo. The Specific Aims of this proposal are: 1. To understand the molecular mechanisms of Hsf4 regulation in mammalian cells. 2. To investigate the function of Hsf4 in animal models. 3. To understand the function of Hsf4 splice variants in animal models. [unreadable] [unreadable]